Double strand breaks (DSBs) are lethal DNA lesions that arise during meiosis, immune system development, and cancer therapy. Ionizing radiation (IR) and some chemotherapeutics kill tumors by inducing DSBs, which can have associated end damage that blocks repair. Repair of damaged ends is important because it shapes the efficiency and safety of cancer therapy. However, previous DSB repair research has focused on undamaged breaks. The proposed research will determine the mechanisms of damaged end repair by nonhomologous end joining (NHEJ), the predominant DSB repair pathway in mammals. The ultimate step in NHEJ is the ligation of ends by DNA Ligase IV (Lig4); I have shown that it is more likely than other pathways to join damaged ends. Lig4 functions only in the context of the NHEJ complex so it is unclear whether damage tolerance is intrinsic to the ligase. I hypothesize that Lig4 is a specialized damaged end ligase, and this activity is important for the response to cancer therapy. I am creating variants of Lig4 that separate function in vitro: they efficiently repair undamaged, but not damaged breaks. I will generate cell lines expressing these Lig4 variants and measure repair of damaged DSB ends. I will expose these cell lines to chemotherapeutic agents and ionizing radiation to determine if Lig4 damaged end repair is important for the response to cancer therapy. I will determine whether NHEJ capacity on damaged ends corresponds to radiosensitivity and chemoresistance in cells. These results will show whether Lig4 damaged end repair is a potentially druggable target to improve existing cancer therapies. Some damaged ends must be processed prior to repair. One processing factor is DNA polymerase mu, which fills in gaps at DSB ends. Surprisingly, pol mu preferentially adds ribonucleotides (RNA), rather than deoxyribonucleotides (DNA), to DNA ends. Preliminary data suggests that these ribonucleotides stimulate Lig4 activity, but it is unclear when and why this happens. I hypothesize that ribonucleotides added by pol mu facilitate Lig4 tolerance of damaged ends. I will identify when ribonucleotides are important for damaged end repair. Then, I will generate variants of Lig4 that abolish its stimulation by ribonucleotides. My results will reveal the biological significance of this startling repair phenomenon which introduces RNA into the genome. Many cancer therapies rely on damaged double strand breaks to kill cancer cells with specificity. The proposed research will provide mechanistic insight into the poorly understood process of damaged DSB repair. We will also identify requirements for damaged DSB repair that can be targeted to improve the effectiveness of cancer therapies.